Department of Orthopaedic Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan; Department of Anatomy and Neuroscience, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan.
Department of Orthopaedic Surgery, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan.
Spine J. 2021 Feb;21(2):343-351. doi: 10.1016/j.spinee.2020.08.011. Epub 2020 Aug 25.
Spinal cord injury (SCI) can lead to increased phosphorylation of p38 in spinal cord microglia. This is one of the main causes for the development of persistent pain. Recently, we reported our study on the activation of p38 mitogen-activated protein kinases (MAPK) in spinal microglia, which has been considered the key molecule for the onset and maintenance of neuropathic pain after peripheral nerve injury, using a rat model. We also reported that the RhoA/Rho-associated coiled-coil containing protein kinase (ROCK) pathway mediates p38 activation in spinal microglia in peripheral nerve injury. But the precise mechanisms of neuropathic pain induced by SCI are still unclear.
This study aimed to examine the activation of microglia and the p38 MAPK expression in the lumbar spinal cord after thoracic SCI in rats, and the correlation to the therapeutic effect of ROCK inhibitor ripasudil in rats with SCI.
Male Sprague-Dawley rats underwent thoracic (T10) spinal cord contusion injury using an Infinite Horizon impactor device. SCI rats received ROCK inhibitor ripasudil (24 nmol/day or 240 nmol/day) from just before SCI to 3 days after SCI.
The mechanical threshold in the rat's hind paws was measured over four weeks. Morphology of microglia and phosphorylation of p38 (p-p38) in the lumbar spinal cord and were analyzed using immunohistochemistry.
The p-p38 positive cell and Iba1 (a maker of microglia) positive area were significantly increased at the lumbar spinal dorsal horn (L4-5) 3 days and 7 days after SCI compared with the sham-control (p<.05), whereas phosphorylated p38 was co-localized with microglia. Three days after SCI, the intensity of phosphorylated p38 and Iba1 immunoreactive cells in the dorsal horn was significantly lower in the ripasudil treated groups than in the saline group. However, administration of ROCK inhibitor did not affect the numbers of microglia. Moreover, the withdrawal threshold of the ripasudil-treated rats was significantly higher than that of the saline-injected rats on 14 days and 28 days after SCI.
Our results suggest that activation of ROCK in spinal cord microglia is likely to have an important role in the activation of p38 MAPK, which has been considered as a key molecule that switches on neuropathic pain after SCI. Inhibition of ROCK signaling may offer a means in developing a novel neuropathic pain treatment after SCI. It may help patients with neuropathic pain after SCI.
The findings in the present study regarding intracellular mechanisms suggest that modulation of ROCK signaling may be a focus for novel treatment for neuropathic pain after SCI.
脊髓损伤（SCI）可导致脊髓小胶质细胞中 p38 的磷酸化增加。这是持续性疼痛发展的主要原因之一。最近，我们报道了我们使用大鼠模型对脊髓小胶质细胞中 p38 丝裂原活化蛋白激酶（MAPK）的激活的研究，该研究被认为是周围神经损伤后神经病理性疼痛发生和维持的关键分子。我们还报道了 RhoA/Rho 相关卷曲螺旋蛋白激酶（ROCK）通路介导周围神经损伤后脊髓小胶质细胞中 p38 的激活。但是，SCI 引起的神经病理性疼痛的确切机制仍不清楚。
本研究旨在检测大鼠胸段 SCI 后腰椎脊髓中小胶质细胞的激活和 p38 MAPK 表达，并探讨 ROCK 抑制剂 ripasudil 对 SCI 大鼠的治疗效果。
雄性 Sprague-Dawley 大鼠使用 Infinite Horizon 撞击器装置造成胸段（T10）脊髓挫伤伤。SCI 大鼠在 SCI 前至 SCI 后 3 天给予 ROCK 抑制剂 ripasudil（24 nmol/天或 240 nmol/天）。
在四周内测量大鼠后爪的机械阈值。使用免疫组织化学分析腰椎脊髓背角（L4-5）中小胶质细胞的形态和磷酸化 p38（p-p38）。
与假对照（p <.05）相比，SCI 后 3 天和 7 天，脊髓背角 L4-5 处 p-p38 阳性细胞和 Iba1（小胶质细胞标志物）阳性面积明显增加，而磷酸化的 p38 与小胶质细胞共定位。与生理盐水组相比，SCI 后 3 天，ripa 处理组背角中磷酸化 p38 和 Iba1 免疫反应细胞的强度明显降低。然而，ROCK 抑制剂的给药并不影响小胶质细胞的数量。此外，在 SCI 后 14 天和 28 天，ripa 处理大鼠的退缩阈值明显高于生理盐水注射大鼠。
我们的结果表明，脊髓小胶质细胞中 ROCK 的激活可能在 p38 MAPK 的激活中起重要作用，p38 MAPK 被认为是 SCI 后神经病理性疼痛发生的关键分子。抑制 ROCK 信号可能为 SCI 后开发新的神经病理性疼痛治疗方法提供一种手段。它可能有助于 SCI 后神经病理性疼痛患者。
本研究关于细胞内机制的发现表明，调节 ROCK 信号可能是 SCI 后神经病理性疼痛治疗的新焦点。
